Week 16 Lecture, Part II P450 Detoxification System Biochemistry of Nutrition, 560B Dr.

Charles Saladino The P450 Cytochrome System This system refers to a unique species of cysteine-thiolate bound heme proteins in bacteria, plants, and animals, and which also represents a huge array of different oxygenases serving a diverse set of compounds (i.e. structurally different substrates). These proteins are encoded by a genetic superfamily of hundreds of genes. The designation P450 is due to the fact that under certain condition the proteins (P) absorb maximally spectrophotometrically at 450 nm. Their substrates are cholesterol, fatty acids, steroids, and a large array of exogenous compounds, referred to collectively as xenobiotics. Xenobiobtics include food additives, pesticides, cigarette smoke, environmental toxins, etc. These can be ingested, inhaled, or absorbed through the skin. I find it quite amazing that our body is able to detoxify so compounds that it has never seen before. The Cytochrome P-450 system is found within the microsomal fraction of the cell (smooth endoplasmic reticulum) and in the inner mitochondrial membrane, primarily in the liver (see below). Although not exclusively, a key feature of this system is the conversion of potentially toxic substances (usually hydrophobic) into less toxic, water-soluble products that can be more easily excreted. Therein lays the secret of the success of the P-450 Detoxification System that is, rendering the compounds more water-soluble and, thus, more easily excreted. This P450 system has far-reaching biomedical implications that include: 1. conversion of chemicals to highly reactive, unwanted molecules that can lead to cellular damage, cell death, mutations, etc. 2. production of steroid hormones. 3. metabolism of fatty acids, prostaglandins, leukotrienes, and retinoids 4. inactivation of therapeutic drugs 5. enzyme inhibition or induction that can result in drug-to-drug interactions and adverse effects. So you get the impression the system not only detoxifies, but it can also convert the good into bad or not good into even worse. Many potential carcinogens become ultimate carcinogens, thanks to the P-450 System. I have included a diagram of some of the reactions some of which are beneficial, some which could be harmful to us.

Basically this detoxification system involves biotransforming compounds by one or a combination of two phases, known as Phase I and Phase II. Phase I uses oxygenase enzymes, which utilize oxygen to form a more water-soluble compound that contains one oxygen atom (often in the form of a hydroxyl group), with the other oxygen atom going to form water. In Phase II any one several polar molecules are conjugated to the compound to be detoxified, again resulting in a more water soluble compound that can be more readily excreted from the body. The groups that can be conjugated to the toxicant include glucuronic acid (look that structure up and find it to be a derivative of glucose), sulfate, an acetyl group, glutathione, water, and amino acids. Sometimes Phase I is all that is necessary, whereas for some toxicants both Phase I and II are required for detoxification.

I should also add the possibility of a Phase III system, which, briefly, involves cells exporting toxins into the blood before the toxicants can do harm. This, in fact, is often seen in cancer cells that become resistant to certain chemotherapies, as well as in bacteria that become resistant to an antibiotic, because in both cases they can actively pump out of their cells the drug before is can kill them. Thus, such a system also believes to be operative in some cells in our body. So now lets add more important specifics. At the top of the reaction table is an equation which summarizes the Phase I reactions, each catalyzed by a monooxygenase of some kind, depending upon the substrate. Notice that NADPH is required as a reducing equivalent (What pathway does that come from?), with the substrate-OH, water, and NADP+ as products. As mentioned above, the two main sites of this detoxification system are located on the inner mitochondrial membrane (where the ETS is located separately from the P450 system, and on the outside of the smooth endoplasmic reticulum. The function of the mitochondrial system is to take part in steroid hydroxylation in hormone producing tissues, including the ovaries, testes, placenta, and adrenal cortex. For example, cholesterol is converted to 20, 22-dihydroxycholesterol by a mixed function oxidase enzyme of the P-450 system. This is then converted to pregnenolone, an important steroid precursor. This system also requires electron-transferring proteins (adrenodoxin and adrenodoxin reductase), the system for cleaving side chains. This system is found in the mitochondria of the adrenal cortex cells, where active steroid synthesis takes place. The microsomal system associated with the membranes of the smooth endoplasmic reticulum (especially in the liver) is designed to handle xenobiotic detoxification, as described above. The above figure illustrates this systems actions, utilizing NADPH. This is the toxicant deactivation system, which unfortunately can convert some pounds to more toxic and even carcinogenic compounds. In fact, often when you hear that a compound is a carcinogen, it might not be in and of itself, but it can become an ultimate carcinogen after it is converted to another molecule by the P450 system. The reactions represented by both the microsomal and mitochondrial systems both require passing electrons along a system similar to, but not at all identical to, the ETS. In the microsomal system, the final electron acceptor is oxygen. However, in both cases, no ATP is produced, as that is not their purpose. Heme proteins are part of this system (working in conjunction with oxygenases, which catalyze oxidation reactions and thus electron shifting), because Fe is a good electron acceptor (now Fe++) and electron donor (now Fe+++). One other set of reactions not mentioned yet is the desaturase system. In this case, saturated fatty acids are desaturated by desaturase enzymes that utilize the NADPH/cytochrome proteins of the P450 system. Again, this is not for detoxification purposes, but it is an important feature of the detox system. This is summarized in the figure below:

Now lets close this discussion with some thoughts about the Phase II system of the detoxification process. To illustrate this action of the P450 system, I will use the example acetaminophen (Tylenol)-induced toxicity in the liver when alcohol is ingested. The figure below will illustrate this. Alcohol is an inducer of and a substrate for CYP2E1, which is a P450 enzyme that can convert acetaminophen to a highly reactive, potentially toxic intermediate normally present in low concentrations. This intermediate is called NAPQI (N-acetyl-p-benzoquinoneimine). When too much acetaminophen is ingested, the levels of NAPQI rise to the point where serious liver damage can occur. Alcohol induces CYP2E1 synthesis, resulting in higher levels of NAPQI. Remember, the CYP2E1 enzyme is a P450 enzyme. Normally, when acetaminophen levels are low, and when there is no alcohol consumption with the drug, there is sufficient glutathione (GSH in the figure) to conjugate with the NAPQI to protect the liver from this metabolite, because it is produced in very small amounts. This is because normally, unless an excess dose of acetaminophen is ingested, the drug is conjugated with glucuronic acid and/or sulfate to produce a non-toxic, water soluble compound that is easily excreted from the body. However, you can see from the figure that high levels of CYP2E1 induced by alcohol can overwhelm the GSH and lead to a very cytotoxic substance that can conjugate with proteins can lead to death of the hepatocytes. Thus, it is suggested that no alcohol be taken during approximately a seven or eight hour period following acetaminophen ingestion. This would give time for the alcohol to be metabolized and thus decrease the levels of the CYP2E1 enzyme. This nicely illustrates the potential of the Phase II P450 system to both detoxify a drug. At the same time, it also demonstrates the capability of the P450 system to cause production of a compound that is potentially quite harmful to the liver.